PROJECT SUMMARY Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance is a growing challenge and methicillin-resistant Staphylococcus aureus (MRSA) infections are more difficult to treat, resulting in increased burden for both patients and healthcare systems. Infections with community-associated methicillin-resistant S. aureus (CA-MRSA) of the USA300 lineage cause especially severe disease in the USA, affecting otherwise healthy individuals and provoking extensive necrosis in skin and lung despite antibiotic treatment. Human polymorphonuclear leukocytes (PMN) dominate the initial innate immune cellular response to invading microorganisms such as S. aureus. Optimal PMN microbicidal action relies on collaboration between oxidants generated by the phagocyte NADPH oxidase and an array of proteins stored in PMN granules. Among phagocytes, PMN are unique, as they possess myeloperoxidase (MPO) in their granules and thus have the singular capacity to oxidize chloride and thereby generate HOCl, a potent microbicide. How S. aureus senses and resists the oxidative microenvironment of the phagosome is unclear. S. aureus primarily perceives extracellular signals using two-component systems (TCS), and we and others have shown the agr quorum-sensing TCS is important for S. aureus survival in PMNs. Our overall goal is to investigate the mechanisms through which S. aureus resists human PMN oxidative killing and their consequences for S. aureus and for PMN. To address these questions, in Specific Aim 1 we will investigate the contribution of the agr system to SPIN regulation and expression. SPIN is a small secreted protein that was recently shown to directly bind to human MPO and prevent H2O2 from entering the active site, and we discovered in preliminary studies that SPIN is regulated by the agr system. We hypothesize that the agr-dependent expression of SPIN is critical for S. aureus survival within human PMN. We will investigate SPIN expression in WT and regulatory mutants, characterize the SPIN gene promoter, and determine the contribution of SPIN to agr-dependent survival of S. aureus within PMN. In Specific Aim 2, we will identify new mechanisms of S. aureus resistance to PMN-oxidative killing. In a rational approach, we inactivated YjiE (ORF 93) in USA300, a conserved HOCl-responsive transcription factor identified in E. coli, and observed increased sensitivity to the PMN-specific oxidant HOCl. Additionally, in preliminary screening, we have discovered USA300 mutant strains that are more resistant and sensitive to HOCl. In this aim we will characterize the YjiE regulon in USA300. We will also continue investigating HOCl resistant strains to identify target genes and finish screening the transposon library for new HOCl-responsive targets. Finally, we will assess the fate of PMN harboring mutant S. aureus strains with differential response to HOCl. Understanding of mechanisms by which ingested S. aureus thwart PMN will provide a framework for creating novel interventions to improve human health.